JP7252280B2 - Novel insulin analogue and use thereof - Google Patents

Description

The present invention provides novel insulin analogues, in particular in vitro compared to native insulin.
In vitro) potency increased insulin analogues and uses thereof.

Insulin is a blood sugar-regulating hormone secreted from the pancreas of the human body. It transfers excess glucose in the blood to the cells, supplies the cells with an energy source, and maintains blood sugar at a normal level. However, in the case of diabetics, the lack of such insulin or the inability of insulin to perform its normal function due to insulin resistance and beta-cell dysfunction. Therefore, diabetics cannot use the glucose in the blood as an energy source, and show hyperglycemia symptoms with high glucose levels in the blood, and eventually excrete sugar in the urine, which can lead to various complications. cause the disease. Therefore, diabetics with abnormal production of insulin (Type I) or due to insulin resistance (Type II) require insulin therapy, and insulin administration can regulate blood glucose to normal levels.

Human insulin is composed of two polypeptide chains, the A and B chains containing 21 and 30 amino acid residues, respectively, which are interconnected by two disulfide bridges. In the case of insulin, unlike other proteins and peptide hormones, its half-life in the body is extremely short, so it does not show a continuous therapeutic effect, and in order to exert its effect, it has to be administered continuously and repeatedly. There is Such frequent dosing causes great pain and inconvenience to the patient, so improvements in patient compliance, safety and convenience are desired.

Here, formulation studies and chemical conjugation of various proteins to enhance the patient's quality of life and therapeutic efficacy by increasing the in vivo half-life and reducing the administration frequency of protein drugs such as insulin. Research on the body is underway.

Insulin is known to remove sugar from the blood by binding to insulin receptors, and the sequence of native insulin can be altered to modulate its potency. The in vivo potency of insulin can be adjusted by substituting amino acids of insulin with other amino acids or deleting specific amino acids, and highly active insulin derivatives exhibit potency equal to or greater than that of natural insulin even in small amounts. can be a very desirable form therapeutically. In particular, amino acid substitutions in the A and/or B chains of insulin have been extensively studied in terms of pharmacokinetic effects of insulin action after subcutaneous injection.

Under such circumstances, the present inventors have made intensive efforts to enhance the action and effect of insulin. The inventors have confirmed that the in vitro potency is remarkably increased compared to insulin and that it can be effectively used for the treatment of diabetes, thus completing the present invention.

Scheit, Nucleotide Analogs, John Wiley, New York, 1980 Uhlman and Peyman, Chemical Reviews, 90: 543-584, 1990

It is an object of the present invention to provide novel insulin analogues, in particular insulin analogues with increased in vitro potency compared to native insulin.

Another object of the present invention is to provide a pharmaceutical composition for treating diabetes comprising the insulin analogue as an active ingredient.

Another object of the present invention is to provide a method of treating diabetes, which comprises administering the insulin analogue or a pharmaceutical composition containing it as an active ingredient to an individual in need thereof.

前記一般式（１）において、
Ｘａａ１は、アラニン、グリシン、グルタミン、ヒスチジン、グルタミン酸またはアスパラギンであり、
Ｘａａ２は、アラニン、グルタミン酸、グルタミン、ヒスチジンまたはアスパラギンであり、
Ｘａａ３は、アラニン、セリン、グルタミン、グルタミン酸、ヒスチジンまたはアスパラギンであり、
Ｘａａ４は、アラニン、チロシン、グルタミン酸、ヒスチジン、リジン、アスパラギン酸またはアスパラギンであり、
Ｘａａ５は、アラニン、ロイシン、チロシン、ヒスチジン、グルタミン酸またはアスパラギンであり、
Ｘａａ６は、アラニン、チロシン、セリン、グルタミン酸、ヒスチジンまたはアスパラギンであり、
Ｘａａ７は、アスパラギン、グリシン、ヒスチジンまたはアラニンである。 As one aspect for achieving the above object, the present invention includes the A chain of SEQ ID NO: 3 represented by the following general formula (1) and the B chain of SEQ ID NO: 4 represented by the following general formula (2) Providing Insulin Analogues:
General formula (1)

In the general formula (1),
Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid or asparagine;
Xaa2 is alanine, glutamic acid, glutamine, histidine or asparagine;
Xaa3 is alanine, serine, glutamine, glutamic acid, histidine or asparagine;
Xaa4 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid or asparagine;
Xaa5 is alanine, leucine, tyrosine, histidine, glutamic acid or asparagine;
Xaa6 is alanine, tyrosine, serine, glutamic acid, histidine or asparagine;
Xaa7 is asparagine, glycine, histidine or alanine.

前記一般式（２）において、
Ｘａａ８は、チロシン、グルタミン酸またはアスパラギン酸であるか、不在であり、
Ｘａａ９は、フェニルアラニンであるか、不在であり、
Ｘａａ１０は、トレオニンであるか、不在であり、
Ｘａａ１１は、プロリン、グルタミン酸またはアスパラギン酸であるか、不在である
（ここで、配列番号１のＡ鎖及び配列番号２のＢ鎖を含むペプチドは除く）。 general formula (2)

In the general formula (2),
Xaa8 is tyrosine, glutamic acid or aspartic acid or is absent;
Xaa9 is phenylalanine or absent;
Xaa10 is threonine or absent;
Xaa11 is proline, glutamic acid, or aspartic acid, or is absent (except for peptides containing the A chain of SEQ ID NO:1 and the B chain of SEQ ID NO:2).

In a more specific embodiment, the peptide comprises the A chain of general formula (1), and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, and Xaa10 is threonine. It is characterized by containing a B chain.

The insulin analogue comprises the A chain of general formula (1), and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, and Xaa10 is absent. and

In another embodiment, in the A chain of SEQ ID NO:3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, and Xaa6 is tyrosine; Xaa7 is asparagine, SEQ ID NO: 4
wherein Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine and Xaa11 is proline.

In another embodiment, in the A chain of SEQ ID NO:3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is asparagine, Xaa5 is leucine, and Xaa6 is tyrosine; Xaa7 is asparagine, the B chain of SEQ ID NO:4 is characterized by Xaa8 being tyrosine, Xaa9 being phenylalanine, Xaa10 being threonine and Xaa11 being proline.

In another embodiment, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, and Xaa6 is tyrosine; Xaa7 is an asparagine, the B chain of SEQ ID NO:4 is characterized by Xaa8 being a tyrosine, Xaa9 being absent, Xaa10 being a threonine and Xaa11 being a proline.

In another embodiment, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is alanine, Xaa5 is leucine, and Xaa6 is tyrosine; Xaa7 is an asparagine, the B chain of SEQ ID NO:4 is characterized by Xaa8 being a glutamic acid, Xaa9 being absent, Xaa10 being a threonine and Xaa11 being a proline.

in other ways,
Is the insulin analogue according to the invention an A chain of SEQ ID NO: 3 in which Xaa4 is glutamic acid and a B chain of SEQ ID NO: 4 in which Xaa9 is phenylalanine;
Xaa4 is asparagine in the A chain of SEQ ID NO:3 and Xaa9 is phenylalanine in the B chain of SEQ ID NO:4;
Xaa4 is glutamic acid in the A chain of SEQ ID NO:3 and Xaa9 is absent in the B chain of SEQ ID NO:4, or
It is characterized by, but not limited to, the A chain of SEQ ID NO: 3 where Xaa4 is alanine, the B chain of SEQ ID NO: 4 where Xaa8 is glutamic acid and Xaa9 is absent.

In another aspect an insulin analogue according to the invention is characterized in that it comprises the amino acid sequence set forth in SEQ ID NO: 16, 18, 20 or 22.

In another aspect, the invention provides nucleic acids encoding said insulin analogues.

In one embodiment, the nucleic acid according to the present invention is characterized by comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19 and 21.

In another aspect, the invention provides a recombinant expression vector comprising said nucleic acid.

In another aspect, the present invention provides a transformant transformed with the recombinant expression vector.

In another aspect, the present invention provides a method of making said insulin analogue comprising the steps of:
a) producing a recombinant expression vector comprising a nucleic acid encoding said insulin analogue peptide;
b) transforming the recombinant expression vector into a host cell to obtain a transformant;
c) culturing the transformant to express the insulin analogue peptide;
d) separating and purifying the expressed insulin analogue peptide;

In another aspect, the present invention provides a pharmaceutical composition for treating diabetes comprising the insulin analogue as an active ingredient and a pharmaceutically acceptable carrier.

In the following, the invention will be described in more detail.

On the other hand, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application are within the scope of the invention. Moreover, the scope of the present invention is not limited by the specific statements set forth below.

The present invention relates to novel insulins, in particular insulin analogues with increased in vitro potency compared to native insulin.

As used herein, the term "insulin analogue" means an analogue in which some amino acids of natural insulin are modified by addition, deletion or substitution. In particular, it includes various analogs that have increased in vitro potency over the native form through said modifications.

As a hormone secreted from the pancreas, natural insulin generally promotes the absorption of intracellular glucose and inhibits the decomposition of fat to regulate blood sugar in the body. Insulin undergoes processing in the form of proinsulin precursors, which have no blood glucose regulating function, into insulin with blood glucose regulating function. Insulin is composed of two polypeptide chains, the A and B chains containing 21 and 30 amino acid residues, respectively, which are interconnected by two disulfide bridges. The A chain and B chain of native insulin contain the amino acid sequences represented by SEQ ID NOs: 1 and 2 below, respectively.

A chain:
Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 1)
B chain:
Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His
-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr (SEQ ID NO: 2)

The insulin according to the present invention is a recombinant insulin analog, but the present invention is not limited thereto and includes all insulins with increased in vitro potency compared to native insulin. Preferably, it includes inverted insulin, insulin variants, insulin fragments, etc., which can be produced not only by genetic recombination methods but also by solid phase methods. , but not limited to.

The insulin analogue according to the present invention is a peptide having an in vivo blood sugar regulating function similar to that of insulin, and such a peptide is an insulin agonist.
, insulin derivative, insulin fragment, insulin variant, etc. are all included.

As used herein, the term "insulin agonist" refers to a substance that binds to insulin receptors in vivo and exhibits the same biological activity as insulin, regardless of the structure of insulin.

In the present invention, the term "insulin derivative" shows homology to each of the amino acid sequences of the A-chain and B-chain of natural insulin, and some groups of amino acid residues are chemically substituted (e.g. α-methylated, α-hydroxylated), deleted (e.g. deaminated) or modified (e.g. N-methylated) forms, meaning peptides that have the function of regulating blood sugar in the body. You may In addition, "insulin derivatives" include peptide mimics and low-molecular-weight or high-molecular-weight compounds that are capable of binding to insulin receptors and regulating blood sugar in the body, even if there is no amino acid sequence homology with natural insulin. may be

As used herein, the term "insulin fragment" refers to a form of insulin in which one or more amino acids have been added or deleted, even if the added amino acids are non-naturally occurring amino acids (e.g., D-form amino acids). Often such insulin fragments have the function of regulating blood sugar in the body.

In the present invention, the term "insulin variant" refers to a peptide that differs from insulin in one or more amino acid sequences and has the function of regulating blood sugar in the body.

The production methods used for the insulin agonists, derivatives, fragments and variants of the present invention may be used independently or in combination. For example, peptides that differ in one or more amino acid sequences, have deamination introduced at the amino terminal amino acid residue, and have the function of regulating blood sugar in vivo are also included in the scope of the present invention.

The insulin analogues according to the present invention have amino acid substitutions, additions, deletions or post-translational modifications (e.g., methylation, acylation, Ubiquitination, intramolecular covalent linkages) are introduced to encompass any peptide that has increased in vitro potency compared to the native form. Atypical or non-naturally occurring amino acids, as well as the 20 amino acids commonly found in human proteins, may be used in the amino acid substitutions or additions. Commercial sources of atypical amino acids include Sigma-
Aldrich, ChemPep and Genzymepharmaceuticals may be included. Peptides containing such amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies such as American peptide company, Bachem, USA, and Anygen, Korea.

Specifically, the insulin analogue according to the present invention includes a modification or deletion of a specific amino acid residue in the A-chain and B-chain of natural insulin as described above, preferably the A-chain of natural insulin. A specific amino acid residue may be modified, and a specific amino acid residue of the B chain may be modified/changed or deleted.

Preferably, the insulin analogues of the present invention have glutamic acid, asparagine or alanine substituted for amino acid tyrosine at position 14 of the A-chain amino acid sequence represented by SEQ ID NO: 1 to increase the in vitro potency of native insulin. Alternatively, tyrosine, which is the 16th amino acid sequence of the B chain amino acid sequence represented by SEQ ID NO: 2, is replaced with glutamic acid, or phenylalanine, which is the 25th amino acid, is deleted, or both may be included. .

前記一般式（１）において、
Ｘａａ１は、アラニン、グリシン、グルタミン、ヒスチジン、グルタミン酸またはアスパラギンであり、
Ｘａａ２は、アラニン、グルタミン酸、グルタミン、ヒスチジンまたはアスパラギンであり、
Ｘａａ３は、アラニン、セリン、グルタミン、グルタミン酸、ヒスチジンまたはアスパラギンであり、
Ｘａａ４は、アラニン、チロシン、グルタミン酸、ヒスチジン、リジン、アスパラギン酸またはアスパラギンであり、
Ｘａａ５は、アラニン、ロイシン、チロシン、ヒスチジン、グルタミン酸またはアスパラギンであり、
Ｘａａ６は、アラニン、チロシン、セリン、グルタミン酸、ヒスチジンまたはアスパラギンであり、
Ｘａａ７は、アスパラギン、グリシン、ヒスチジンまたはアラニンである。 More preferably, the insulin analogue according to the present invention is a novel peptide comprising the A chain of SEQ ID NO:3 represented by the following general formula (1) and the B chain of SEQ ID NO:4 represented by the following general formula (2): may be:
General formula (1)

In the general formula (1),
Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid or asparagine;
Xaa2 is alanine, glutamic acid, glutamine, histidine or asparagine;
Xaa3 is alanine, serine, glutamine, glutamic acid, histidine or asparagine;
Xaa4 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid or asparagine;
Xaa5 is alanine, leucine, tyrosine, histidine, glutamic acid or asparagine;
Xaa6 is alanine, tyrosine, serine, glutamic acid, histidine or asparagine;
Xaa7 is asparagine, glycine, histidine or alanine.

前記一般式（２）において、
Ｘａａ８は、チロシン、グルタミン酸またはアスパラギン酸であるか、不在であり、
Ｘａａ９は、フェニルアラニンであるか、不在であり、
Ｘａａ１０は、トレオニンであるか、不在であり、
Ｘａａ１１は、プロリン、グルタミン酸またはアスパラギン酸であるか、不在である。 general formula (2)

In the general formula (2),
Xaa8 is tyrosine, glutamic acid or aspartic acid or is absent;
Xaa9 is phenylalanine or absent;
Xaa10 is threonine or absent;
Xaa11 is proline, glutamate or aspartate or absent.

Here, peptides containing the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2 may be omitted.

In one more specific aspect,
In the general formula (1),
Xaa1 is glycine;
Xaa2 is glutamine;
Xaa3 is serine;
Xaa4 is alanine, glutamic acid or asparagine;
Xaa5 is Leucine,
Xaa6 is tyrosine;
Xaa7 is asparagine;
In the general formula (2),
Xaa8 is tyrosine or glutamic acid;
Xaa9 is phenylalanine or absent;
Xaa10 is threonine,
Xaa11 can be, but is not limited to, an insulin analogue peptide that is proline, glutamic acid, or aspartic acid, or is absent.

In one more specific aspect,
In the general formula (1),
Xaa1 is glycine;
Xaa2 is glutamine;
Xaa3 is serine;
Xaa4 is glutamic acid or asparagine;
Xaa5 is Leucine,
Xaa6 is tyrosine;
Xaa7 is asparagine;
In the general formula (2),
Xaa8 is tyrosine;
Xaa9 is phenylalanine or absent;
Xaa10 is threonine,
Xaa11 can be, but is not limited to, an insulin analogue peptide that is proline, glutamic acid, or aspartic acid, or is absent.

In one more specific embodiment, the peptide comprises the A chain of general formula (1), and the B chain of SEQ ID NO: 4, wherein Xaa8 is tyrosine, Xaa9 is absent, and Xaa10 is threonine. , Xaa11 may contain, but are not limited to, a B chain that is proline, glutamic acid, or aspartic acid, or is absent.

The insulin analogue comprises the A chain of general formula (1) above, and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is absent, and Xaa11 is proline, glutamic acid or asparagine. It may include, but is not limited to, a B chain that is acid or absent.

The insulin analogue is Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, Xaa6 is tyrosine, Xaa7 in the A chain of SEQ ID NO:3. is asparagine, in the B chain of SEQ ID NO: 4 Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, Xaa11 is proline, glutamic acid or aspartic acid, or may be absent, It is not limited to this.

The insulin analogue is Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is asparagine, Xaa5 is leucine, Xaa6 is tyrosine, Xaa7 in the A chain of SEQ ID NO:3. is asparagine, in the B chain of SEQ ID NO: 4 Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, Xaa11 is proline, glutamic acid or aspartic acid, or may be absent, It is not limited to this.

In another embodiment, in the A chain of said SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, Xaa6 is tyrosine, Xaa7 is asparagine, in the B chain of SEQ ID NO: 4 Xaa8 is tyrosine, Xaa9 is absent, Xaa10 is threonine, Xaa11 is proline, glutamic acid or aspartic acid, or may be absent, It is not limited to this.

In another embodiment, in the A chain of said SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is alanine, Xaa5 is leucine, Xaa6 is tyrosine, Xaa7 is asparagine, in the B chain of SEQ ID NO: 4 Xaa8 is glutamic acid, Xaa9 is absent, Xaa10 is threonine, Xaa11 is proline, glutamic acid or aspartic acid, or may be absent, It is not limited to this.

According to one preferred embodiment, insulin analogues according to the invention comprise:

i) insulin analogue 1: a peptide in which the 14th amino acid of the A-chain amino acid sequence represented by SEQ ID NO:3 is glutamic acid and the 25th amino acid of the B-chain amino acid sequence represented by SEQ ID NO:4 is phenylalanine, It has the amino acid sequence set forth in SEQ ID NO:16 and is encoded by a nucleic acid having the base sequence set forth in SEQ ID NO:15.

ii) insulin analogue 2: a peptide in which the 14th amino acid of the A-chain amino acid sequence represented by SEQ ID NO:3 is asparagine and the 25th amino acid of the B-chain amino acid sequence represented by SEQ ID NO:4 is phenylalanine, It has the amino acid sequence set forth in SEQ ID NO:18 and is encoded by a nucleic acid having the base sequence set forth in SEQ ID NO:17.

iii) insulin analogue 3: a peptide in which the 14th amino acid in the A chain amino acid sequence represented by SEQ ID NO: 3 is glutamic acid and the 25th amino acid in the B chain amino acid sequence represented by SEQ ID NO: 4 is deleted, It has the amino acid sequence set forth in SEQ ID NO:20 and is encoded by a nucleic acid having the base sequence set forth in SEQ ID NO:19.

iv) insulin analogue 4: 14th amino acid in the A chain amino acid sequence represented by SEQ ID NO: 3 is alanine, 16th amino acid in the B chain amino acid sequence represented by SEQ ID NO: 4 is glutamic acid, and 25th amino acid is deleted and has the amino acid sequence set forth in SEQ ID NO:22, which is encoded from a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:21.

The term "in vitro effect" as used herein in relation to insulin analogues is
It means the glucose uptake of insulin analogues, and in the present invention, the E
It is expressed by the result of measuring _C50 .

In a preferred embodiment of the invention, the in vitro efficacy of insulin analogues 1 to 3 according to the invention was determined based on the above method, resulting in 238.4% for insulin analogue 1 and 241% for insulin analogue 2 compared to native insulin. It was confirmed that the insulin analogue according to the present invention exhibited a significantly increased in vitro potency of 2 to 7 times compared to native insulin (Table 1). 1).

In another aspect, the invention provides nucleic acids encoding said insulin analogues.

As used herein, the term "nucleic acid" refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) present in single-stranded or double-stranded form, including genomic DNA, cDNA and RNA transcribed therefrom. Nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues with modified sugar or base sites (Non-Patent Documents 1 and 2). Nucleic acids of the invention can be isolated or produced using standard molecular biology techniques. For example, it can be amplified via PCR (polymerase chain reaction) from the native insulin gene sequence (NM_000207.2, NCBI) using appropriate primer sequences and standard synthetic techniques using automated DNA synthesizers. can be manufactured using

The nucleic acids of the present invention preferably contain the nucleotide sequences set forth in SEQ ID NOs: 15, 17, 19 and 21. The present invention provides not only the nucleotide sequences described in SEQ ID NOS: 15, 17, 19 and 21, but also 70% or more homology, preferably 80% or more homology, more preferably 90% or more homology with the above sequences. more preferably 95% or more homology, and most preferably 98% or more homology, wherein the peptide substantially encoded by the nucleic acid binds to insulin's in vivo receptor and behaves like insulin. including any that exhibit significant biological activity.

The term "homology" as used in the present invention indicates the degree of similarity between the amino acid sequence of a wild type protein or the base sequence encoding it, and the amino acid sequence of the present invention or It includes sequences that have more than a percent sequence identity with the base sequence as described above. Such homology can be determined by comparing two sequences with the naked eye, but can also be determined using a bioinformatic algorithm that aligns the sequences to be compared and analyzes the degree of homology. may Homology between the two amino acid sequences may be expressed as a percentage. A useful automated algorithm is the Wisconsin Genetic
s Software Package (Genetics Computer Group, Madison, W., USA) with GAP, BESTFIT, FASTA and TFASTA computer software modules. The automated alignment algorithms of the module include alignment algorithms of Needleman & Wunsch, Pearson & Lipman and Smith & Waterman alignments. Algorithms and homology determinations for other useful sequences have been automated with software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.

In another aspect, the present invention provides a recombinant vector comprising a nucleic acid encoding said insulin analogue. The recombinant vector according to the present invention may typically be constructed as a vector for cloning or as a vector for expression, and may be constructed using prokaryotic or eukaryotic cells as host cells.

As used herein, the term "vector" refers to a recombinant vector capable of expressing a protein of interest in a suitable host cell, the nucleic acid containing the essential regulatory elements operably linked so that the nucleic acid insert is expressed. means construct. In the present invention, a recombinant vector containing a nucleic acid encoding an insulin analogue can be produced, and the insulin of the present invention can be produced by transforming or transfecting a host cell with the recombinant vector. Analog may be acquired.

A nucleic acid encoding an insulin analogue in the present invention is operably linked to a promoter. As used herein, the term "operatively linked" refers to a functional sequence between a nucleic acid expression regulatory sequence (e.g., promoter, signal sequence, ribosome binding site, transcription termination sequence, etc.) and another nucleic acid sequence. binding, whereby said regulatory sequence modulates the transcription and/or decoding of said other nucleic acid sequence.

As used herein, the term "promoter" refers to an unencoded nucleic acid sequence upstream of the coding region that contains the binding site for the polymerase and has the activity of initiating transcription into the mRNA of the gene downstream of the promoter, i.e., to which the polymerase binds. A DNA region that initiates transcription of a gene, and is located 5' of the mRNA transcription initiation site.

For example, when the vector of the present invention is a recombinant vector and the host is a prokaryotic cell, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter , rac5 promoter, amp promoter, recA promoter, SP
6 promoter, trp promoter and T7 promoter), a ribosome binding site for initiation of decoding and a transcription/decoding termination sequence.

Vectors that may be used in the present invention include plasmids commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61
, pLAFR1, pHV14, pGEX series, pET series, pPICZα series, pUC19, etc.), phage (e.g., λgt4 λB, λ-Charon, λΔ
z1 and M13) or viruses (eg, SV40, etc.).

On the other hand, when the vector of the present invention is a recombinant vector and the host is a eukaryotic cell, a promoter derived from a mammalian cell genome (e.g., metallothionein promoter) or a mammalian virus-derived promoter (e.g., adenovirus) can be used. late promoter, vaccine virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) may be used, and polyadenylation sequences as transcription termination sequences (e.g., bovine growth hormone terminator and SV40-derived polyadenylation sequences). ) in general.

In addition, the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selectable marker, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. may be used.

The recombinant vector of the present invention may optionally further comprise other sequences to facilitate the purification of the recovered protein of interest, ie single-chain insulin analogues, proinsulin or insulin analogues. Said further optionally included sequence may be a tag sequence for protein purification, for example glutathione S-transferase (Pharmacia
, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and six histidines (hexahistidine), etc., but these examples limit the types of sequences required for purification of the target protein. not a thing

A fusion protein expressed by a recombinant vector containing a tag sequence as described above may be purified by affinity chromatography. For example, if a glutathione-S-transferase was fused, the enzyme's substrate, glutathione, could be used, and if a six histidine tag was used, a Ni-NTA column could be used to determine the desired target. of protein may be readily recovered.

In another aspect, the present invention provides a transformant transformed with a recombinant vector containing a nucleic acid encoding the insulin analogue.

As used herein, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA becomes replicable as a chromosomal element or through completion of chromosomal integration, thereby transforming the external DNA into the cell. It means a phenomenon in which a genetic change is artificially caused by introduction.

Any transformation method may be used for the transformation method of the present invention, and it may be easily carried out by a conventional method in the art. In general, transformation methods include _CaCl2 precipitation, Hanahan method in which _CaCl2 precipitation is enhanced by using a reducing substance called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation. method, protoplasmic fusion method, agitation method using silicon carbide fibers, Agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/
and suppression-mediated transformation methods.

Methods for transforming a recombinant vector containing a nucleic acid encoding an insulin analogue according to the present invention are not limited to the above examples, and transformation or transfection methods commonly used in the art can be used without limitation. good too.

A transformant of the present invention may be obtained by introducing a recombinant vector containing a nucleic acid encoding an insulin analogue, which is a nucleic acid of interest, into a host cell.

A host suitable for the present invention is not particularly limited as long as the nucleic acid of the present invention can be expressed. Particular examples of hosts that may be used in the present invention include Escherichia bacteria such as E. coli; Bacillus bacteria such as Bacillus subtilis; Pseudomonas Pseudomonas bacteria such as Pseudomonas putida; yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; Spodoptera frugiperda ( SF9)
and animal cells such as CHO, COS, BSC, and the like. Preferably, E. coli is used as the host cell.

In a preferred embodiment of the invention, the nucleic acid sequences encoding the insulin analogues 1-4 according to the invention, respectively, were amplified by PCR (polymerase chain reaction) and then the amplified gene fragments were cloned into the pET22b vector (Novagen). . In order to express the insulin analogue in the form of intracellular inclusion bodies, the pET22b vector was treated with the restriction enzymes NdeI and BamHI to remove the signal sequence, and the insulin analogue PCR amplification product was isolated by treatment with the same restriction enzymes NdeI and BamHI. The individual DNAs were inserted into the pET22b cloning vector using T4 DNA ligase. The resulting expression vectors were named pET22b-insulin analogues 1-4, respectively.

The expression vectors pET22b-insulin analogues 1-4 encode the amino acid sequences of SEQ ID NOS: 16, 18, 20 and 22, respectively, under the control of the T7 promoter, and express the respective insulin analogue peptides in the form of inclusion bodies in host cells. expressed.

Recombinant vectors pET22b-insulin analogues 1 to 4 containing nucleic acids encoding insulin analogues of SEQ ID NOS: 16, 18, 20 and 22 were transformed into E. coli to obtain transformants expressing these in the form of inclusion bodies. bottom.

In another aspect, the present invention provides a method for producing an insulin analogue using the transformant.

Preferably, the method according to the invention comprises
a) producing a recombinant expression vector containing a nucleic acid encoding an insulin analogue;
b) transforming the recombinant expression vector into a host cell to obtain a transformant;
c) culturing the transformant to express the insulin analogue; and d) isolating and purifying the expressed insulin analogue.

The medium used for culturing transformants in the present invention should meet the requirements of host cell culture in an appropriate manner. The carbon source that may be contained in the medium for the growth of host cells may be appropriately selected according to the judgment of those skilled in the art according to the type of transformant produced, and the culture period and amount may be adjusted. Suitable culture conditions may be employed for this purpose.

Sugar sources that may be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid. , stearic acid, fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These substances may be used individually or as mixtures.

Nitrogen sources that may be used include peptones, yeast extract, gravy, malt extract, corn steep liquor, soybeans, wheat, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. is included. Nitrogen sources may also be used individually or as a mixture.

Phosphorus sources that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain metal salts such as magnesium sulfate or iron sulfate necessary for growth.

Finally, in addition to said substances, essential growth substances such as amino acids and vitamins may be used. Appropriate precursors may also be used in the culture medium. The raw materials mentioned above may be added batchwise or continuously to the culture during cultivation by a suitable method. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture. Antifoaming agents such as fatty acid polyglycol esters may also be used to suppress bubble formation. Oxygen or an oxygen-containing gas (eg, air) is injected into the culture to maintain aerobic conditions.

Cultivation of the transformant according to the present invention is usually carried out at a temperature of 20°C to 45°C, preferably 25°C to 40°C. Cultivation is also continued until maximum production of the desired insulin analogue is obtained, and for such purposes the culture may typically last from 10 to 160 hours.

As described above, the transformants of the present invention will produce insulin analogues by composing suitable culture conditions according to the host cells, and peptides produced based on the vector construction and host cell characteristics. -N-glycosidases may be secreted into the host cell cytoplasm, into the periplasmic space or extracellularly.

Proteins expressed in or out of host cells may be purified by conventional methods. Examples of purification methods include salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (e.g., protein fraction precipitation, etc. using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, reverse Techniques such as chromatography, such as phase column chromatography, and ultrafiltration, alone or in combination, may be applied.

The transformant of the present invention is characterized in that the recombinant vector pET22b-insulin analogues 1-3 introduced into it expresses the insulin analogues 1-3 in the form of inclusion bodies under the control of the T7 promoter. Therefore, it is desirable to convert the insulin analogues 1 to 3 expressed in the form of inclusion bodies into a soluble form by an appropriate method, followed by separation and purification.

A preferred embodiment of the invention may further comprise the following steps for isolating and purifying the insulin analogue expressed in the form of inclusion bodies from the transformant.

d-1) Harvesting and disrupting transformant cells from the culture medium;
d-2) recovering and refolding the expressed insulin analogue peptide from the disrupted cell lysate;
d-3) purifying the refolded insulin analogue peptide by cation exchange chromatography;
d-4) treating the purified insulin analogue peptide with trypsin and carboxypeptidase B;
d-5) Purifying the treated insulin analogue peptide sequentially by cation exchange chromatography and anion exchange chromatography.

In another aspect, the present invention provides a pharmaceutical composition for treating diabetes comprising said insulin analogue.

A pharmaceutical composition containing an insulin analogue according to the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes and flavoring agents used for oral administration. Also, in the case of injections, buffers, preservatives, soothing agents, solubilizers, tonicity agents, stabilizers, etc. may be mixed and used. Bases, excipients, lubricants, preservatives and the like may be used. Various dosage forms of the pharmaceutical composition of the present invention may be prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups and wafers, and injections may be prepared in unit dosage ampules or multiple dosage forms. may In addition, it may be formulated as solutions, suspensions, tablets, pills, capsules, sustained-release preparations, and the like.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginic acid, gelatin, calcium phosphate, calcium silicate. , cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil and the like may be used. It may also contain fillers, anti-aggregating agents, lubricants, wetting agents, perfumes, emulsifiers, preservatives and the like.

In another aspect, the present invention provides a method of treating diabetes comprising administering said insulin analogue or a pharmaceutical composition comprising the same to an individual in need thereof.

Since the insulin analogue according to the present invention exhibits significantly increased in vitro potency compared to native insulin, diabetes can be treated by administering the insulin analogue or a pharmaceutical composition containing the same.

In the present invention, "administration" means introducing a given substance into a patient by any suitable method, and the route of administration of said conjugate can be any general route as long as the drug can reach the target tissue. It may also be administered via any route. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration may be used, but are not limited to these. However, since peptides are digested when administered orally, it is desirable to coat the active agent in the composition for oral use or formulate it so as to protect it from decomposition in the stomach. It may preferably be administered in the form of an injection. Also, the pharmaceutical compositions may be administered by any device capable of transferring the active ingredients to the target cells.

In addition, the pharmaceutical composition of the present invention may be selected depending on the type of drug as an active ingredient, together with some related factors such as the disease to be treated, administration route, patient age, sex, body weight and severity of disease. determined accordingly. Since the pharmaceutical composition of the present invention has excellent in vivo persistence and potency, the number and frequency of administration of the pharmaceutical composition of the present invention can be significantly reduced.

Since the insulin analogue according to the present invention exhibits markedly increased in vitro potency compared to native insulin, a sufficient therapeutic effect can be expected even with administration of a small amount, and the insulin analogue is very useful for the treatment of diabetes.

Fig. 2 shows the result of analyzing the purity of insulin analogues according to the present invention by protein electrophoresis, representative of insulin analogue 1. Figs. Lane 1: Size marker Lane 2: Insulin analogue 1 1 shows the result of analyzing the purity of insulin analogues according to the present invention by reversed-phase chromatography and size exclusion chromatography, representatively for insulin analogue 1. FIG. Fig. 2 shows peptide mapping analysis results of insulin analogues according to the present invention, representative of insulin analogue 1; "USP insulin" in FIG. 3 means native insulin used as a control group.

The present invention will now be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1 Production of Insulin Analogue Expression Vector Direction for Amplification of Insulin Analogues into which the A-chain and/or B-chain A- and/or B-chain of Native Insulin are Altered in Amino Acid Variation After synthesizing a pair of reverse primers, PCR was performed using the proinsulin cDNA as a template. At this time, proinsulin cDNA (SC128255, OriGene) (reference sequences: BC005255.1 and AAH05255) was used as a template in pET22b vector (Novagen).
For smooth recombinant expression of insulin, a N-terminal fusion partner is added to the N-terminus of the cloned proinsulin cDNA.
), the base sequence of SEQ ID NO: 23 (ATG GCA ACA ACA TCA ACA GCA ACT ACG CGT) encoding the amino acid sequence of Met Ala Thr Thr Ser Thr Thr Ala Thr Thr Thr Arg (SEQ ID NO: 24) was inserted.

Specifically, in the present invention, insulin analogues containing amino acid sequence variations as shown in Table 1 below were synthesized.

In Table 1, insulin analogue 1 contains a substitution of tyrosine, which is the 14th amino acid in the A-chain amino acid sequence of natural insulin represented by SEQ ID NO: 1, with glutamic acid;
Insulin analogue 2 contains a substitution of asparagine for tyrosine, which is the 14th amino acid in the A-chain amino acid sequence of native insulin represented by SEQ ID NO: 1,
Insulin analogue 3 is a substitution of tyrosine, which is the 14th amino acid in the natural insulin A chain amino acid sequence represented by SEQ ID NO: 1, with glutamic acid and the natural insulin B chain amino acid sequence represented by SEQ ID NO: 2. It contains a deletion of the 25th amino acid, phenylalanine.

Insulin analog 4 is a substitution of tyrosine, which is the 14th amino acid in the natural insulin A chain amino acid sequence represented by SEQ ID NO: 1, with alanine and the natural insulin B chain amino acid sequence represented by SEQ ID NO: 2. It contains a substitution of glutamic acid for tyrosine, the 16th amino acid, and a deletion of the 25th amino acid, phenylalanine.

The respective forward and reverse primer pairs designed for amplification of the insulin analogues 1-3 are shown in Table 2 below.

In Table 2, the primer pair of SEQ ID NOs: 5 and 6 is for substituting glutamic acid for tyrosine, which is the 14th amino acid in the A chain amino acid sequence of natural insulin, and the primer pair of SEQ ID NOs: 7 and 8. is for substituting asparagine for the 14th amino acid tyrosine in the natural insulin A chain amino acid sequence, and the primer pair of SEQ ID NOs: 9 and 10 is for the 25th amino acid sequence in the natural insulin B chain amino acid sequence. The primer pair of SEQ ID NOS: 11 and 12 is for substituting alanine for the 14th amino acid tyrosine in the A chain amino acid sequence of natural insulin. , and the primer pair of SEQ ID NOs: 13 and 14 replaces tyrosine, which is the 16th amino acid in the B-chain amino acid sequence of natural insulin, with glutamic acid.

PCR for amplification of insulin analogues containing the relevant variants was performed using 150 ng of template DNA,
1 ml of each 100 pM primer, 5 ml of 2.5 mM dNTP, 10 units of pfx polymerase (Invitrogen, USA) and 10× buffer were mixed to prepare a reaction solution,
After initial denaturation at 95° C. for 30 seconds, annealing at 95° C. for 30 seconds, 55° C. for 30 seconds and 68° C. for 6 minutes was repeated 18 times, and finally reacted at 68° C. for 5 minutes. The PCR amplified product obtained from this was extracted using a gel extraction kit (Qiagen, Germany) and then treated with restriction enzymes NdeI and BamHI to prepare an insert. The pET22b vector (Novagen, USA) was subsequently cut with the same restriction enzymes and extracted using the same gel extraction kit. The inserts were ligated into the vector prepared in this manner using T4 ligase to produce expression vectors pET22b-insulin analogues 1-4. The expression vector contains a nucleic acid encoding the amino acid sequences of insulin analogues 1-4 under the control of the T7 promoter, and is capable of expressing the insulin analogue protein in the form of inclusion bodies in the host.

The expression vector pET22b-insulin analogue 1 according to the present invention obtained therefrom comprises a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 15, which encodes an insulin analogue having the amino acid sequence set forth in SEQ ID NO: 16, Expression vector pET22b-insulin analogue 2 comprises a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 17, which encodes an insulin analogue having the amino acid sequence set forth in SEQ ID NO: 18, and expression vector pET22b-insulin analogue 3 contains a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 19, which encodes an insulin analogue having the amino acid sequence set forth in SEQ ID NO: 20, and an expression vector pET22b-insulin analogue 4 set forth in SEQ ID NO: 21 which encodes an insulin analogue having the amino acid sequence set forth in SEQ ID NO:22.

Table 3 below shows the DNA and protein sequences of each insulin analog 1-4.

Example 2 Expression of Recombinant Insulin Analogues Recombinant expression of insulin analogues according to the invention under the control of the T7 promoter was carried out as follows. Escherichia coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB ^- mB ^- ) gal λDE3) (Novagen, USA) was transformed with each of the insulin analogue expression vectors prepared in Example 1 above. Transformation was performed according to the method recommended by Novagen, the manufacturer of said E. coli BL21-DE3. A single colony transformed with an insulin analogue expression vector was picked, inoculated into 2×LB (Luria Broth, LB) medium containing 50 μg/ml ampicillin, and cultured at 37° C. for 15 hours. Mix the recombinant strain culture medium and 2xLB medium containing 30% glycerol at a ratio of 1:1 (v/v), dispense 1 ml each into cryo-tubes, and store at -140°C. kept. This was used as a cell stock for recombinant production of insulin analogues according to the invention.

For recombinant expression of insulin analogues according to the invention, 1 vial of each cell stock was thawed, inoculated into 500 ml of 2xLB medium and cultured with shaking at 37°C for 14-16 hours. When the _OD600 value was 5.0 or higher, the culture was terminated and used as a seed culture medium. Using a 50 L fermentor (MSJ-U2, BE MARUBISHI, Japan), the seed culture was inoculated into 17 L of fermentation medium to initiate initial bath fermentation. The culture conditions are a temperature of 37°C and an air volume of 20 L/min (1
vvm), and the agitation speed was set at 500 rpm and the pH was maintained at 6.7 using 30% aqueous ammonia. Fermentation progress was carried out in fed-batch culture by adding an additional feeding solution when nutrients in the culture were limited. Strain growth was monitored by OD value and IPTG (isopropyl-beta-D-thiogalactoside) was introduced at a final concentration of 500 μM when the OD value reached 100 or higher. Cultivation was further advanced for about 23-25 hours after introduction of IPTG, and after culturing was completed, recombinant strains were harvested using a centrifuge and stored at -80°C until further use.

Example 3 Isolation and Purification of Recombinant Insulin Analogues In order to isolate and purify the recombinant insulin analogues expressed from the transformants in Example 2 above, the insulin analogues expressed in the form of insoluble inclusion bodies were first separated into soluble forms. , the cells were disrupted and refolded as described below.

<3-1> Recovery and Refolding of Recombinant Insulin Analogs
After resuspension in is-HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl and 0.5% Triton X-100), microfluidizer processor M-110EH ( 15,000 ps using AC Technology Corp. Model M1475C)
The cells were crushed by applying a pressure of i. The disrupted cell lysate was centrifuged at 7,000 rpm for 20 min at 4° C., the supernatant was discarded, and 3 L of wash buffer (0.5% Triton X-100 and 50 mM Tris-HCl, pH 8.0) was added. ), 0.2 M NaCl, 1 mM EDTA). After centrifugation at 7,000 rpm for 20 minutes at 4° C., the pellet was resuspended in distilled water and centrifuged in the same manner. A pellet was taken from this and resuspended in 400 ml of buffer (1 M glycine, 3.78 g cysteine-HCl, pH 10.6) and stirred at ambient temperature for 1 hour. To recover the resuspended recombinant insulin analogue, 400 ml of 8 M urea was added and then stirred at 40° C. for 1 hour. After centrifugation at 7,000 rpm for 30 min at 4° C. for refolding of the solubilized recombinant insulin analogue, the supernatant was taken, where 7.2 L of distilled water was added to the peristaltic pump. pump) at a flow rate of 1000 ml/hour and stirred at 4° C. for 16 hours.

<3-2> Purification by cation exchange chromatography A cation exchange column (Source S, GE Healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol was applied to the cation exchange column (Source S, GE Healthcare). 3-1> was loaded and allowed to bind. Subsequently, the protein of the insulin analogue was subjected to a linear concentration gradient of 10× column volume so that the concentration was 0%→100% using 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol. was eluted from the column.

<3-3> Treatment with trypsin and carbopeptidase B Salts were removed from the sample eluted with the desalting column in Example <3-2>, and a buffer solution (10 mM Tris-HCl, pH 8.0) was added. 0). After adding trypsin corresponding to 1000 molar ratio and carboxypeptidase B corresponding to 2000 molar ratio to the protein amount of the obtained sample, the mixture was stirred at 16° C. for 16 hours. The reaction was terminated by lowering the pH to 3.5 using 1M sodium citrate (pH 2.0).

<3-4> Purification by Cation Exchange Chromatography The sample whose reaction was terminated in Example <3-3> was equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol. The column (Source S, GE Healthcare) was reloaded and bound. followed by potassium chloride 0.5M and 45%
The insulin analogue protein was eluted from the column with a 10× column volume linear concentration gradient from 0% to 100% using 20 mM sodium citrate buffer (pH 2.0) containing ethanol.

<3-5> Purification by Anion Exchange Chromatography Salts were removed from the sample eluted with the desalting column in Example <3-4> and exchanged with a buffer solution (10 mM Tris-HCl, pH 7.5). To separate the pure insulin analogues in the samples obtained from this, they were loaded and bound to an anion exchange column (Source Q, GE Healthcare) equilibrated with 10 mM Tris buffer (pH 7.5). Subsequently, the insulin analog protein was eluted from the column with a 10× column volume linear concentration gradient from 0% to 100% using 10 mM Tris buffer (pH 7.5) containing 0.5 M sodium chloride. .

Purity of the purified insulin analogues was analyzed using protein electrophoresis (SDS-PAGE) and reversed phase and size exclusion chromatography, the results of which are shown in Figures 1 and 2, respectively. In addition, amino acid variations were confirmed through peptide mapping and molecular weight analysis of each peak, which is shown in FIG.

As a result, it was confirmed that the amino acid sequences were modified according to the purpose of each insulin analogue.

Example 4 Comparison of In Vitro Potency of Native Insulin and Insulin Analogs To determine the in vitro potency of the insulin analogs isolated and purified in Example 3 above, the 3T3-L1 cell line derived from mice was differentiated into adipocytes. A glucose uptake (or lipid synthesis capacity) test was performed using 3T3-L1 cell line (ATCC, CL-173) was passaged 2-3 times a week using DMEM (Dulbeco's Modified Eagle's Medium, Gibco, Cat.No, 12430) medium containing 10% NBCS (newborn calf serum). cultured and maintained. 3T3
- The L1 cell line was suspended in a medium for differentiation (DMEM containing 10% FBS), seeded in a 48-well plate at 5 x ¹⁰⁴ cells per well, and cultured at 37°C for 48 hours. 1 μg/ml human insulin (Sigma, Cat.
No. I9278), 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma, Cat. No. I5879), and 1 μM dexamethasone (Dexamethasone, Sigma, Cat. No. D4902) were mixed, and the existing medium was removed. After that, 250 μl was added per well. After 48 hours, the medium for differentiation was replaced with a medium supplemented with only 1 μg/ml human insulin. After that, it was confirmed that differentiation into adipocytes was induced for 7 to 9 days while exchanging the medium for differentiation with 1 μg/ml human insulin every 48 hours.

For the glucose absorption test, the adipocyte-differentiated cells were washed once with serum-free DMEM medium, and 250 μl of each medium was added to induce serum starvation for 4 hours.

Human insulin and insulin analogues were prepared by 10-fold serial dilutions from 5 μM to 0.005 nM in serum-free DMEM medium. 250 μl each of the prepared insulin samples was added to the cells and incubated for 24 hours at 37° C., 5% CO ₂ incubator. In order to measure the residual amount of glucose in the cultured medium, 200 μl of the medium was taken, diluted 5-fold with D-PBS, and subjected to GOPOD analysis (GOPOD Assay Kit, Megazyme, Megazyme, Cat. No. K- GLUC). Based on the absorbance of the standard glucose solution, the concentration of glucose in the rest of the medium was converted, and the _EC50 of the glucose absorption capacity of each insulin analogue was calculated and shown in Table 4 below.

As shown in Table 4, insulin analogue 1 according to the invention increased glucose by 238.4%, insulin analogue 2 by 241.7% and insulin analogue 3 by 705% compared to native human insulin. Absorptive capacity was observed.

From the above results, it was confirmed that the in vitro potency of the insulin analogue according to the present invention was greatly increased by 2 to 7 times compared to native insulin, which means that the insulin analogue is effective in blood in an actual living body. It suggests that the half-life is greatly increased and that it can be provided as a stable insulin formulation, and that it can be effectively used as a therapeutic drug for diabetes.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention is not the above detailed description, but the meaning and scope of the following claims, and all modifications or variations derived from the equivalent concepts thereof. should be interpreted as

Claims (9)

An insulin analogue peptide comprising the A chain of SEQ ID NO: 3 represented by the following general formula (1) and the B chain of SEQ ID NO: 4 represented by the following general formula (2):
General formula (1)

general formula (2)

Here, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is aspartic acid, Xaa5 is leucine, Xaa6 is tyrosine, and Xaa7 is asparagine,
In the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, and Xaa11 is proline. A nucleic acid encoding the insulin analogue peptide of claim 1 . A recombinant expression vector comprising the nucleic acid of claim 2 . A transformant transformed with the recombinant expression vector according to claim 3 . 5. The transformant according to claim 4 , wherein said transformant is E. coli. A method for producing an insulin analogue peptide according to claim 1, comprising the steps of:
a) producing a recombinant expression vector comprising a nucleic acid encoding the insulin analogue peptide of claim 1;
b) transforming a host cell with said recombinant expression vector to obtain a transformant;
c) culturing said transformant to express the insulin analogue peptide; and d) isolating and purifying the expressed insulin analogue peptide. 7. The method of claim 6 , wherein said host cell is E. coli. The separation and purification are
d-1) Harvesting and disrupting transformant cells from the culture medium;
d-2) recovering and refolding the expressed insulin analogue peptide from the disrupted cell lysate;
d-3) purifying the refolded insulin analogue peptide by cation exchange chromatography;
d-4) treating the purified insulin analogue peptide with trypsin and carboxypeptidase B; and d-5) sequentially purifying the treated insulin analogue peptide by cation exchange chromatography and anion exchange chromatography. 7. The method of claim 6 , comprising the step of: A pharmaceutical composition for treating diabetes, comprising the insulin analogue peptide of claim 1 as an active ingredient and a pharmaceutically acceptable carrier.

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